Acellular matrix grafts: preparation and use

ABSTRACT

Acellular matrix grafts are provided with are isolated from natural sources and consist essentially of a collagen and elastin matrix which is devoid of cellular components. The grafts are useful scaffolds which promote the regeneration of muscle tissue and aid in restoring muscle function. Due to their acellular nature, the grafts lack antigenicity. As a result, the acellular matrix grafts can be isolated from autographic, allographic or xenographic tissues.

This invention was made with Government support under Grant No. DK51101,awarded by the National Institutes of Health. The Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Bladder reconstruction plays an essential role in the treatment ofvoiding disorders characterized by low bladder capacity or highintravesical pressures or both-1 The ideal material should bebiocompatible and mechanically reliable, resist extraluminal infection,deter or tolerate intraluminal infection, and be easy to implantsurgically. The material should preserve renal function, provideadequate urinary storage at low pressure and allow volitional, completeevacuation of urine per urethram. To achieve this goal, autoaugmentationtechniques and a great variety of synthetic and naturally derivedbiomaterials have been used.

Synthetic materials have been unsuccessful because of foreign-bodyreactions, resulting in stone formation, collapse, infection, rejection,or extrusion and migration of the graft (see Barrett, et al. Semin.Urol. 2:167-75 (1984); Bohne, et al. J. Urol. 77:725-732 (1957);Stanley, et al. J. Urol. 107:783-787 (1972); Swinney, et al. Br. J.Urol. 33:414-429 (1961)). As a result, synthetic materials have beenused primarily as temporary implants to allow bladder regeneration tooccur (see Taguchi, et al. J. Urol. 108:752-756 (1977); Tsuji, et al. J.Urol. 97:1021-1028 (1967)). However, the majority of studies confirmregeneration of transitional cell epithelial lining on the inner surfaceof the graft without adequate reconstruction of a functional detrusormuscle. Natural materials for bladder reconstruction have mostlyretracted with time (see, Baret, et al. Surg. Gynec. Obstet. 97:633-639(1953); Kelami, J. Urol. 105:518-22 (1971)) and the alloplastic totalbladder prosthesis is still at an investigational stage in animals.

Autoaugmentation by enterocystoplasty with either small bowel or colonhas well-documented urodynamic benefits. Sidi, et al. J. Urol.136:1201-4 (1986). Because of complications, including metabolicacidosis (McDougal, J. Urol. 147:1199-208 (1992)) rupture (Bauer, et al.J. Urol. 148:699-703 (1992)), mucus production, chronic bacteriuria,stone formation (Golomb, et al. Urology 34:329-38 (1989)), and thepotential for osteoporosis and malignancy (Filmer, et al. J. Urol.143:671 (1990)), the search for other suitable materials continues.Gastrocystoplasty circumvents some of these problems, but peptic ulcersand perforations, the hematuria/dysuria syndrome, and metabolicalkalosis negate some of its potential advantages over intestinalsegments. Mitchell, et al. Oxford, Blackwell Scientific, pp 439-444(1993). Recently the technique for enterocystoplasty lined withurothelium has been shown to increase bladder capacity while takingadvantage of the inert properties of an intact urothelial lining. Buson,et al. Urology 44:743-748 (1994); Gonzales, et al. Urology 45:124-129(1995). Gastrointestinal segments in general have proven to enhancebladder capacity and compliance, thus protecting the upper tract andrenal function. Unfortunately, they are unable to support normalmicturition, which often necessitates clean intermittent catheterizationor other supportive measures to ensure complete bladder evacuation.

To overcome this functional shortcoming, natural and/or biodegradablematerials serving as a scaffold for the ingrowth of host bladder wallcomponents have been tried with encouraging results. Atala, et al. J.Urol. 150:608-12 (1993); Knapp, et al. J. Endourol. 8:125-30 (1994);Novick, et al. J. Biomed. Mater. Res. 12:125-47 (1978); Scott, et al.Br. J. Urol. 62:26-31 (1988). The bladder wall tissue thus regeneratedhas shown the potential to provide functional augmentation in terms ofan enlargement of the bladder without compromising its voidingabilities.

Despite the above efforts, there remains a need for new materials whichare useful for grafting by serving as a scaffold for the development ofnew muscle tissue. The scaffolding material should be antigenic andcapable of use in a variety of organs and individual hosts.Surprisingly, the present invention provides such materials and furtherprovides methods for their preparation and use.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides an acellular matrix graftwhich is isolated from muscle tissue, and consists essentially ofacellular collagen and elastin. The muscle tissue which is the source ofthe graft is, for example, bladder tissue or other smooth muscle tissuesuch as heart tissue or ureter or urethra tissue.

In another aspect, the present invention provides methods of preparingacellular matrix grafts in which muscle or nerve tissue is isolated andfreed from cells and cellular components by mechanical, chemical orenzymatic methods, or by combinations of mechanical, chemical andenzymatic methods to leave a scaffold or graft which is essentiallycollagen and elastin fibers. For example, a bladder acellular matrixgraft can be prepared by:

(a) removing mucosa from an excised bladder cap to provide a bladderwall;

(b) treating the bladder wall with chemical and enzyme agents to releaseintracellular components from the bladder wall to provide anintermediate matrix; and

(c) solubilizing and removing cell membranes and intracellular lipidsfrom the intermediate matrix to provide a bladder acellular matrix graftwhich consists essentially of acellular collagen and elastin.

In yet another aspect, the present invention provides methods ofrestoring muscle function in animals having damaged or diseased muscles.In these methods, the damaged or diseased tissue is removed and replacedwith an organ-specific acellular matrix graft. The surrounding tissuethen grows and infiltrates the scaffold or graft such that muscle tissueis regenerated and muscle function is restored.

In one preferred embodiment, the method is directed to restoring bladderfunction in an animal having a partially damaged bladder, the methodcomprising:

(a) removing the portion of the bladder which is damaged; and

(b) replacing the removed portion with a bladder acellular matrix graftto promote regeneration of bladder tissue and restore the bladderfunction.

In still another aspect, the present invention provides methods forpromoting regrowth and healing of damaged or diseased muscle tissues,said method comprising replacing the damaged or diseased muscle tissuewith an acellular matrix graft which consists essentially of acellularcollagen and elastin, and is prepared from organ-specific tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the working steps for processing a native rat bladderinto a bladder acellular matrix graft (BAMG).

FIG. 2 shows the scanning. electron microscopy of the dog, hamster, andrabbit BAMG at 20× and 5,000× magnification.

FIG. 3 shows the recordings of electrical field stimulation in a hamsterBAMG regenerate and a corresponding host bladder smooth muscle strip 4months after grafting.

DETAILED DESCRIPTION

The following abbreviations are used herein: DMEM, Dulbecco's modifiedEagle's medium; RPMI, Roswell Park Memorial Institute media; HBSS,Hank's Balanced Salt Solution; FCS, fetal calf serum; EGF, epidermalgrowth factor; TGF-β, β-transforming growth factor; H&E, hematoxylin andeosin; PGP, peptide growth protein; DMSO, dimethylsulfoxide;

As used herein the term “allographic tissue” refers to tissue which isisolated from an individual and used in another individual of the samespecies. The term “xenographic tissue” refers to tissue which isisolated from an individual of one species and placed in an individualof another species. The term “autographic tissue” refers to tissueisolated from an individual which is grafted back into that individual.

The term “enzymatic digestion” refers to the degradation of tissuesusing enzymes such as nucleases. Typically, enzymatic digestion will beused to lyse cells and cellular components and remove the lysed productsfrom the surrounding scaffolding of collagen and elastin.

The term “host” refers to an animal which is the recipient of tissuewhich has been purified, cultured and transplanted from another species(the donor) or from itself.

The term “acellular” in the context of a matrix refers to a compositionthat is essentially free of intact cells such that any remaining cellsare not statistically or biologically meaningful in the ability of thematrix to perform as a regenerative support and is free of any unwantedcells that could include antigens which might contribute to rejection ofthe matrix by the immune system of the animal in which the matrix isintroduced.

The term “xenograph or xenographic” refers to between species such asbetween rat and dog or pig and human.

The phrase “consisting essentially of” in the context of matrixcompositions refers to a composition that includes elastin and collagenbut does not include chemicals or naturally occurring compositions orcells which are derived from the source of origin of the matrix and thatstatistically or significantly contribute or improve the ability of thecomposition to perform as a substrate for regenerating cells; but, thephrase does include exogenously added incredients that substantially orsignificantly alter the grafting process as it relates to cell growthrates apart from the performance of the matrix. For example, theaddition of growth factors to the matrix would still be included in themeaning of the phrase because the added ingredients act upon theinvading cells directly and apart from the matrix and such factors donot act upon the matrix itself. Similarly, antibiotics would not alterthe matrix performance but may indirectly improve its success rate byinhibiting growth of contaminating bacteria.

General

Ideal materials for muscle replacement (e.g., partial bladderreplacement) should possess good physicochemical properties andmechanical reliability as well as biocompatibility. Bowel is mostcommonly used in various procedures of urinary diversion and neobladderreplacement for the treatment of bladder cancer, neurogenic bladderdysfunction, bladder exstrophy, and interstitial cystitis. However, itsuse is not without long-term complications. See, for example,Bunyaratavey, et al. J. Med. Assoc. Thai., 76:327 (1993) and Khoury, etal. Urology, 40:9 (1992). For this reason, the search for differentmaterials and techniques for functional bladder replacement has longbeen the subject of a number of investigations.

Naturally derived materials for bladder wall substitution, such aslyophilized dura, have often been limited by their retraction over time(Kelami, J. Urol., 105:518 (1971)) and the alloplastic replacement ofthe urinary bladder is still at an investigational stage in animals(Rohrmann, et al. J. Urol., 156:2094 (1996)). Previous research hasdemonstrated that collagen-based materials, such as porcine smallintestinal submucosa (SIS) (Knapp, et al. J. Endourol., 8:125 (1994))and polymer scaffolds with urothelial and smooth muscle cells (Yoo, etal. J. Urol., 155:338 (1996)), have the best potential in terms of theirregenerative capability and functional capacity. A new biomaterial, thebladder acellular matrix graft (BAMG) described in detail below and inProbst, et al. Brit. J. Urol., 79:505 (1997), has recently been reportedto be successful in rats for partial bladder replacement. The BAMG hasnow been demonstrated to provide complete regeneration of all wallcomponents, and in vitro studies of matrix-regenerated muscle stripsshow approximately 50% contractility when compared with host strips(see, Piechota, et al. Urol. Res., 25:2.12A (1997)). Because a BAMG canderive from mechanically, chemically and enzymatically processed nativebladders of any species, a BAMG can be used as a homologous or xenogenicgraft. Additionally, the acellularity of this graft material results indecreased antigenicity. Further provided herein is a demonstration thatBAMG-regenerated rat detrusor smooth muscle actively contributes to invivo storage and voiding while preserving the low-pressure reservoirfunction of the bladder. Still further, the contractility of theBAMG-regenerated bladders has been demonstrated with in vitro electricaland pharmacologic stimulation techniques.

Description of the Embodiments

In one aspect, the present invention provides a matrix graft consistingessentially of collagen and elastin. Preferably, the graft is anacellular matrix graft which is isolated from muscle tissue and whichconsists essentially of acellular collagen and elastin. The graftcomponents (collagen and elastin) form a scaffold for the ingrowth ofhost components such as smooth muscle, blood vessels, and nerves. Theacellular matrix graft can be isolated from a variety of sources such asbladder tissue, heart tissue, or ureter or urethra tissue and typicallyindicates essentially no cell nuclei when stained with dyes such as, forexample, trichrome, H&E, α-actin and PGP. Preferred sources will dependon the anticipated use of the graft. For example, a preferred graft forbladder augmentation will be bladder tissue, which can be autographic,allographic or xenographic tissue. The most preferred sources of thematrix graft are those hosts which yield grafts of similar constructionto the target recipient (considering, for example, the ratio of collagento elastin fibers and the types of collagen fibers). Particularlysuitable sources include tissues from rats, hampsters, dogs, pigs,rabbits, bovine and humans.

In a particularly preferred embodiment, the acellular matrix graft is abladder acellular matrix graft (BAMG) which is isolated from rat,rabbit, hampster, dog, pig or human bladder tissue. As above, the graftis an acellular matrix consisting essentially of acellular collagen andelastin, and indicates essentially no cell nuclei when stained withtrichrome, H&E, α-actin, or PGP dyes. In one preferred embodiment, thematrix graft is isolated from human bladder tissue and has an elasticmodulus of about 0.40 to about 0.80 MPa. In another preferredembodiment, the matrix graft is isolated from rat bladder tissue and hasan elastic modulus of about 0.80 to about 2.10 MPa. In yet anotherpreferred embodiment, the matrix graft is isolated from pig bladdertissue and has an elastic modulus of about 0.25 to about 0.60 MPa.

The acellular matrix grafts can be prepared using known techniques forsurgical removal of the source tissue and subsequent treatment of thetissue to remove cells and cell contents including cell membranes andintracellular lipids. FIG. 1 provides a schematic representation of thesteps used in processing a rat bladder dome into a rat BAMG. In brief,the bladder dome is first surgically removed and inverted. Mucosa isremoved by, for example, peeling or scraping, and the remaining laminapropria and muscularis is soaked in successive solutions of bufferedpreservatives, antibiotics, nucleases and detergents, e.g., sodiumazide, DNAse, sodium deoxycholate and neomycin sulfate. The solutionsserve to remove cellular material and preserve the BAMG until grafting.While the methods below are described with reference to bladder tissue,one of skill in the art will understand that similar steps can beapplied to the isolation and preparation of acellular matrix grafts fromother tissues and sources.

Accordingly, in another aspect, the present invention provides a methodfor the preparation of a bladder acellular matrix graft, comprising:

(a) removing mucosa from an excised bladder cap to provide a bladderwall;

(b) treating the bladder wall with chemical and enzyme agents to releaseintracellular components from the bladder wall to provide anintermediate matrix; and

(c) solubilizing and removing cell membranes and intracellular lipidsfrom the intermediate matrix to provide a bladder acellular matrixgraft.

In this aspect of the invention, mucosa is removed from an excisedbladder cap. The bladder cap can be isolated from the sources indicatedabove, and preserved for later use, or it can be isolated just prior toprocessing. Removal of mucosa from the excised bladder cap willtypically be carried out by first treating the tissue with sodium azidein PBS, then using manual techniques including, for example, gentlescraping with a suitable instrument or a glass slide. Other methods canalso be used to remove the mucosa, including submucosa using similarmethods but without peeling off the mucosa.

The resultant muscular wall which remains following removal of themucosa is then treated with a combination of chemical and enzyme agentsto initiate cell lysis thereby releasing cellular components andinitiating the removal of cells from the matrix. A number of chemicaland enzyme agents are known to be useful for effecting cell lysis.Suitable chemical agents include sodium azide, sodium chloride, sodiumphosphate and potassium chloride. Typically, chemical treatment iscarried out in a buffered aqueous solution (e.g., HEPES-buffered saline(HBS) or 1 M NaCl, 10 mM phosphate buffer) for a period of time of fromabout 1 to about 24 hours, more preferably about 3 to about 10 hours andmost preferably about 5 to about 6 hours. Enzyme agents which are usefulin this aspect of the invention include nucleases such as, for example,DNase. Following cell lysis with the chemical and enzyme agents, anintermediate matrix is obtained.

The intermediate matrix is then treated to solubilize and remove anyremaining cell membranes and intracellular lipids, thereby producing anacellular matrix graft. The treatments used to solubilize the cellmembranes and intracellular lipids are typically chemical methods. Inpreferred embodiments, the chemical methods use a sodium desoxycholatesolution containing sodium azide. More preferably, the solubilizingmethods use an aqueous solution of from about 1% to about 8%desoxycholate, containing of from about 0.05% to about 5% sodium azide.In a particularly preferred embodiment, the cell membranes andintracellular lipids are removed from the intermediate matrix using anaqueous solution of about 4% sodium desoxycholate containing about 0.1 %sodium azide. The period of time necessary to solubilize the cellmembranes and intracellular lipids will depend on the temperature atwhich solubilization is carried out and the identity and concentrationof the solubilizing agents. Typically, the solubilization is carried outat room temperature for a period of from about 1 to about 24 hours, morepreferably about 3 to about 10 hours and most preferably about 5 toabout 6 hours.

The resultant acellular matrix graft is typically washed with aphysiologically compatible buffer (e.g., PBS) and used immediately, orit can be treated with a preservative (e.g., 0.1% sodium azide and 10%neomycin sulfate) and stored at about 4° C. until ready for use. One ofskill in the art will appreciate that the processing steps describedabove for bladder tissue can be applied equally to the isolated andprocessing of other smooth muscle tissues such as urethra or ureter.

In yet another aspect, the present invention provides a method forpromoting regrowth and healing of damaged or diseased muscle tissues. Inthis method, damaged or diseased muscle tissue is replaced with anacellular matrix graft prepared as described above. Accordingly, thediseased or damaged muscle tissue can be initially removed usingsurgical procedures, and an acellular matrix graft can be incorporatedin its place. The removal of damaged or diseased muscle tissues can beaccomplished using well-known surgical techniques such as, for example,partial cystectomy, augmentation cystoplasty, urethral stricture orureter stricture. The replacement graft will typically be held in placeusing, for example, absorbable sutures, nonabsorbable sutures, orcombinations of the two. Preferably, the sutures used are absorbablesutures.

An appropriate acellular matrix graft is typically one which has similarconstituents and mechanical properties as the host tissue. The matrixgraft can be xenographic, allographic or autographic. In order to moreclosely match the host tissues, allographic or autographic matrix graftsare preferred. When xenographic tissues are used for the preparation ofan acellular matrix graft, the preferred origin of the acellular graftwill be one which provides the nearest approximation to the recipient ofcollagen and elastin. The nearest approximation, or match, can bedetermined by subjecting the graft tissue to a variety of mechanicalproperty tests.

To determine certain mechanical properties, matrix strips and a controlare subjected to physical stress, using a system designed to apply aprecise and reproducible load. Stress/strain curves can be generated(see Examples) and analyzed to describe the physical properties of thematerials. For the bladder acellular matrix graft, the passiveproperties of strain, stress and elastic modulus are similar in the BAMGand the control bladder strips in all species (pig, human and rat weretested). Different strain values in the rat are due to the high amountof collagen type I and the lower amount of elastic fibers than in thepig and human. The tendency of the elastic modulus to be higher in theBAMG strips and the less balanced incline of the correspondingstress/strain graphs is a result of smooth muscle and urothelium loss.It has been noted in previous studies that age-related changes incollagen composition as well as structure (non-enzymatic glycosylationand increased crosslinking) may cause stiffening of the bladder wall(see Cerami, et al. Sci. Am., 256:90 (1987); Monnier, et al. Proc. Natl.Acad. Sci. USA, 81:583 (1984); and Longhurst, et al. J. Urol., 148:1615(1992)).

Regeneration of the muscle tissue supported by the matrix grafttypically includes angiogenesis, smooth muscle growth, and nerveproliferation. Angiogenesis, broadly defined as the growth of newcapillary blood vessels from extant vascular beds, has been proposed tobe regulated by a proliferative and/or morphogenetic pathway (type Icollagen as a template for endothelial cell migration and lumenformation). Sage, H. E. et al. J. Hypertens., 12:S145 (1994). Smoothmuscle growth may originate from the edges of the graft or frompericytes after capillary neovascularization. Tilton, J. Electron.Microsc. Tech., 19:327 (1991). Baskin, et al. (Baskin, et al., J. Urol.,156:1820 (1996)) reported that both intact bladder urothelium andisolated bladder mesenchyme recombined with bladder urothelium from ratfetuses demonstrated expression of smooth muscle differentiation whengrafted under the renal capsule. In contrast, bladder mesenchyme alonefailed to induce smooth muscle expression when grafted. Additionally,the development of a functional extracellular matrix seems to beinfluenced by mechanical forces. The importance of an intact collagenscaffold has been underscored by the finding that physical strainstimulates collagen types I and III expression in bladder urothelial andsmooth muscle cells. Baskin, et al., J. Urol., 150:601 (1993)). Althoughcollagen type I promotes cell migration, differentiation and tissuemorphogenesis during development (Brenner, et al., J. Lab. Clin. Med.,124:755 (1994)), its main function in connective tissues has beenassumed to be withstanding tension (see, Montes, Cell Biol. Int., 20:15(1996)). Collagen type III is thought to be responsible for structuralmaintenance in expansible organs. The histology of the matrix graftspresented demonstrates different features in texture. However, althoughdifferent quantities of collagen types I and III have been shown, bothtypes have been found as major components of the BAMG.

Because active function of the bladder seems mainly to be generated bysmooth muscle cells, the tensile properties have been assumed to beinfluenced by the extracellular matrix. Pasquali-Ronchetti, I. et al.:Ultrastructure of elastin. In: Ciba Foundation Symposium on themolecular biology and pathology of elastic tissues, New York: John Wiley& Sons, pp 31-42, 1995. Elastic fibers are most abundant in tissuessubject to stretching, such as blood vessels, lung, skin and elasticcartilage (Viidik, A. et al. Biorheology, 19:437, 1982), while thecollagen network seems to be responsible for the quality of high tensilestrength in connective tissues. Jain, M. K. et al. Biomaterials, 11:465,1990.

Xenotransplantation of the matrix graft can show variations in thedegree and quality of smooth muscle regeneration (see, Piechota, H. J.et al. Urol. Res., 25:2.12A, 1997). When hamster and pig BAMGs aregrafted to rat bladder, regeneration is best facilitated with theclosest possible structural match between the matrix graft and the hostbladder wall. This has been confirmed by the differences in the purematrix structures presented herein. The similar performance of the pigBAMG with the human BAMG allows the use of pig BAMG material for partialbladder replacement in humans.

Accordingly, in still another aspect, the present invention provides amethod of restoring bladder function in an animal having a partiallydamaged bladder, the method comprising:

(a) removing the portion of the bladder which is damaged; and

(b) replacing the damaged portion with a bladder acellular matrix graftto promote regeneration of bladder tissue and restore the bladderfunction.

In this method, the animal which is being treated can be a rat, pig,dog, or human. Typically, the bladder acellular matrix graft isprepared, as described above, from xenographic or allographic tissue.

The present invention further provides methods of restoring musclefunction in an animal having a partially damaged muscle, the methodcomprising:

(a) removing the portion of the muscle which is damaged; and

(b) replacing the damaged portion with an acellular matrix graft topromote regeneration of muscle tissue and restore the muscle function.

In this method, as in the methods above, the animal which is beingtreated can be a rat, pig, dog, or human. Typically, the acellularmatrix graft is prepared, as described above, from xenographic orallographic tissue. Preferably, the acellular matrix graft is prepared,as described above, from organ-specific xenographic or allographictissue. For example, repairing heart muscle in a human will preferablybe accomplished with an acellular matrix graft from heart tissue of adonor which is preferably human or another mammalian species whichprovides a matrix graft similar in composition to a human heartacellular matrix graft.

The following experimental results are offered by way of example and arenot meant to limit the scope of the invention.

EXAMPLES Example 1

This example illustrates the preparation and characterization of bladderacellular matrix grafts from rat, pig and human bladders.

1.1 Bladder Isolation

Urinary bladders without the trigone were harvested from fresh rat(female, age 5 months, N=20) and pig (female, age 9 months, N=3) and afemale human cadaver (age 65 years). The pig and human bladders werelongitudinally bisected; half was assigned for matrix preparation, andthe other for control. The rat bladders were not bisected; 10 wereassigned to each group.

1. 2 Acellular Bladder Matrix Preparation

In the matrix preparation process, urinary bladders were placed in Petridishes containing 50 mL of 10 mM phosphate-buffered saline (PBS, pH 7.0)and 0.1% sodium azide. The bladders were inverted and the mucosa wasscraped off with a pair of glass slides. The remaining lamina propriaand detrusor muscle were treated with 50 mL of 10 mM PBS—0.1 % sodiumazide and stirred for 5-6 hours to obtain partial cell lysis. Bladderswere then washed with 40 mL of PBS before treatment with 50 mL of 1 Msodium chloride containing 2000 Kunitz units DNase (Sigma; St. Louis,Mo.) and then stirred for 12-14 hours; this step was repeated 2 to 4times depending on the source of bladder. With this, cell lysis wascomplete and all the intracellular components were released. The sampleswere then treated with 50 mL of 4% sodium deoxycholate containing 0.1 %sodium azide and stirred for 5-6 hours to solubilize the lipid bilayercell membranes and intracellular membrane lipids; this step was repeatedtwice. The resultant bladder acellular matrix graft was washed threetimes with 50 mL PBS and stored in 0.1 % sodium azide and 10% neomycinsulfate at 4° C.

1.3 Light Microscopy

Acellular graft specimens were fixed in 10% buffered formalin for atleast 24 hours. After dehydration in graded ethanol, the specimens wereembedded in paraffin (sections: 5 μm) and stained with hematoxylin andeosin (H&E) for nuclei and α-actin for smooth muscle. These studiesshowed the structures of the matrices (rat, pig and human) to be anacellular scaffold and thus confirmed the effectiveness of the matrixpreparation process. The elastic fibers were stained according to Hart'stechnique. see, Luna, MANUAL OF HISTOLOGIC STAINING METHODS OF THE ARMEDFORCES INSTITUTE OF PATHOLOGY, 3rd ed. New York: McGraw Hill Book Co.,pp 79-80 (1968). Collagen types were identified as reported by Junqueiraet al., Cell Tissue Res., 202:453 (1979). Histologic sections werestained with Sirius red and viewed under polarized light (thick fibers[type I] appear strongly birefringent yellow or red, and thin fibers[type III] appear greenish and are weakly birefringent). Elastic fiberswere seen in the bladder matrix of all three types, and histologicdifferences were apparent in both elastin and collagen fibers. In thepig, and particularly the human matrix, the number of elastic fibers wasgreater than in the rat. Picrosirius-stained specimens under thepolarizing microscope demonstrated that type I was the major collagenfiber in the rat bladder matrix. In contrast, type III fibers wereabundant in the pig and human bladder matrix. The ratio of thick to thincollagen fibers in the rat BAMG was notably higher than in the pig orhuman BAMG.

1.4 Transmission Electron Micrcoscopy

Specimens were immersed in a fixative (2.5% glutaraldehyde and 2.5%paraformaldehyde) in 0.15 M sodium cacodylate buffer. After primaryfixation, the samples were placed in a drop of fixative on dental waxand cut in 3 mm segments. Specimens were post-fixed in 2% osmiumtetroxide, block-stained in tannic acid, and dehydrated in a gradedseries of ethanol and propylene oxide, after which they were embedded inresin. Thin sections (500 Å) were cut and mounted on 200-mesh coppergrids. After staining in uranyl acetate and lead citrate, examinationwith a Philips 400 Model electron microscope was carried out.

Electron microscopic studies disclosed variations in the structure ofthe acellular matrix scaffold in the different species and supportedlight microscopic findings. There were notably fewer elastic fibers inthe rat bladder matrix than in the pig and human matrix, and theyappeared to be more densely stained. Collagen fibers appeared to beclosely packed with marked variations in diameter in the rat matrix; inthe pig, and particularly the human, they appeared as a loose network.Electron microscopy was not able to distinguish distinct collagen types.

1.5 Strip Preparation and Mechanical Properties

Specimens were assigned to six groups (N=10 in each): viz., the normalbladder and the acellular matrix of each of the three species.Longitudinal strips were obtained from each group. A sandpaper frame wasconstructed around the specimens to facilitate uniform gripping duringtensile testing. The ends of each strip were attached to the smooth sideof the sandpaper with cyanoacrylate adhesive to prevent slippage fromthe clamps. Subsequently, another sandpaper frame was attached to form asandwich around the strip with a window over the test section. Specimenswere wrapped in saline-moistened gauze, covered in plastic wrap, andstored at −20° C.

Before testing, specimens were removed from the freezer and allowed tothaw while immersed in physiologic saline at room temperature. Bothlength and width of the specimens were measured with Verner calipers(±0.02 mm). The width of the specimen was also measured within theframe. The frames were mounted in the grips of a servohydraulic materialtesting machine (MTS Bionix 858, Eden Prairie, Minn., USA). Once mountedin the grips, the sides of the sandpaper frame were cut. The specimenswere distracted longitudinally at a rate of 0.3 mm/sec to failure.Specimen load and grip travel were continuously measured throughout thetests with a precision force transducer (Sensotec, Model 31, Columbus,Ohio) and the test system Linear Variable Differential Transformer(LVDT).

Stress/strain curves for each specimen were generated and the ultimatetensile strength, maximum strain and elastic modulus were determined.The strength (MPa) was calculated by dividing the failure load by thecross-sectional area of the specimen. The maximum strain, i.e. thestrain value corresponding to the ultimate strength, was calculated asthe displacement of the specimen divided by the initial gauge length (inmm/mm). The elastic modulus (MPa) of the strips was defined as the slopeof the most linear region of the stress/strain curve. Photographs ofeach test were taken to analyze the failure patterns of each specimen.Data are given as mean±S.D. Differences between the means of the BAMGand control urinary bladder were tested for statistical significancewith Student's t-test. A value of p≦0.05 was considered significant.

The mechanical properties of the rat, pig and human BAMG and controlurinary bladder strips are summarized in Table 1.

TABLE 1 Tensile Properties of the BAMG versus the Normal Urinary Bladderin Rat, Pig and Human Ultimate Ultimate Tensile Tensile Elastic StrengthStrength Modulus Material No. (mm/mm) (MPa) (MPa) Rat BAMG 10  0.73 ±0.23* 0.68 ± 0.21  1.43 ± 0.59* Rat Bladder 10 2.03 ± 0.44 0.72 ± 0.210.76 ± 0.44 Pig BAMG 10 1.86 ± 0.51 0.29 ± 0.05 0.40 ± 0.13 Pig Bladder10 1.66 ± 0.31 0.32 ± 0.10 0.26 ± 0.18 Human BAMG 10 0.91 ± 0.08 0.13 ±0.05 0.60 ± 0.21 Human Bladder 10 0.69 ± 0.17 0.27 ± 0.14 0.25 ± 0.18Data are given as mean ± S.D. *p < 0.05 vs. normal rat bladder

There was no evidence of specimen slippage within the grids during thetesting procedure. The site of failure during testing varied anddepended on the architecture of the particular specimen, near the endsin some cases and within the center of the strip in others.

Typical stress/strain plots for the BAMG and the control bladder stripsdemonstrated an initial nonlinear region, followed by a linear region,and finally a region where failure occurred in all species. The inclineof the BAMG graphs was less balanced than that of the control bladderstrips in all species.

The mean maximum strain for the BAMG and the control bladder strips wasnot significantly different, except in the rat (p<0.05). Also there wasno significant difference in the ultimate strength between the BAMG andthe control strips in all species (p>0.05); however, the ultimatetensile strength of both the rat bladder and the rat BAMG was higherthan that of the pig and human groups.

The comparison of the elastic modulus for the BAMG and the controlbladder strips did not show any significant difference in either the pigor human specimens (p>0.05), although, in general, the BAMG had a highervalue than the control. In contrast, the elastic modulus of the rat BAMGwas significantly higher than the value for the control rat bladderstrips (p<0.05). The elastic moduli for both the rat bladder and the ratBAMG were higher than those for the pig and human groups.

The biomechanical properties of the newly developed bladder acellularmatrix graft support its use for bladder replacement. Variations of thematrix structure in different species indicate that the closest possiblestructural match between the matrix graft and the host bladder wall willachieve the best functional results.

Example 2

This example illustrates the decreased antigenicity of the bladderacellular matrix graft (AMG) through xenotransplantation and alsoevaluates the in vivo and in vitro functional properties of theBAMG-regenerated rat urinary bladder.

2.1 Preparation of the Bladder Acellular Matrix Graft (BAMG)

Bladders from male Syrian hamsters, male New Zealand white rabbits, andmale mongrel dogs were obtained from our institutions' tissue-sharingprogram. The bladders were excised and treated essentially as describedin Example 1. The hamster BAMGs were used as full-bladder-size grafts,whereas rabbit and dog BAMGs were cut to smaller patches ofapproximately 5×5 mm before grafting.

Histologic sections were examined by light and scanning electronmicroscopy before grafting to confirm the BAMGs' acellularity and thusthe effectiveness of the matrix preparation process. These studiesrevealed an intact framework of collagen and elastin fibers with noevidence of remaining cells, nuclei or other cell fragments. Thestructure, density and thickness of the fibers differed considerablyamong the three BAMG types. The average fiber diameter seemed toincrease with the size of the BAMG donor species; fiber density appearedto be less in the rabbit and dog BAMGs than in the hamster BAMG (FIG.2).

2.2 Surgical Technique

Male and female Sprague Dawley rats (n=20) were anesthetized withpentobarbital (40 mg/kg i.p.) and placed supine on a warmed operatingtable (37° C.). Through a lower midline incision, the bladder wasexposed and a 70% cystectomy was performed without coagulation orligation of the vessels. The previously prepared BAMG was grafted to theremaining host bladder with a continuous polyfilament-coated 8-0 Vicryl™(Ethicon Inc., Somerville N.J.) absorbable suture for both the anteriorand posterior walls. Four nonabsorbable monofilament 7-0 Dermalon™(Davis+Geck, Manati PR) button sutures were placed—one each at theanterior, posterior, left and right sides—to identify the matrixborders. The control animals (n=10) underwent partial cystectomy only.Three 7-0 Dermalon™ button sutures were placed at the left, mid andright sides for later identification of the site of cystectomy.

The hamster, rabbit and dog BAMGs were grafted to 20 Sprague-Dawley rats(the distribution of male to female was 3 to 2 within each group). Fiveanimals died postoperatively (3 male, 2 female). The 3 male rats (2hamster and 1 rabbit BAMG) died within 2-4 days from uremia consequentto urinary extravasation into the abdominal cavity. All 3 presented withcomplete obstruction of the bladder neck and proximal urethra by astaghom stone-like plug formed of coagula and defurfurated fibers of theBAMG (non-mineral material, as shown by both polarizing light microscopyand scanning electron microscopy). Leakage always occurred at the siteof anastomosis. A rupture of the graft itself was never seen. The 2female animals (1 rabbit and 1 dog BAMG) died 3 weeks after surgery froma Corona virus infection associated with severe respiratory tractobstruction and rapid weight loss. A total of 15 grafted rats wereavailable for further evaluation.

Partial cystectomy was performed in 10 control rats (5 male/5 female).None of the animals died of causes related to the surgical procedureitself. However, 3 rats died within 2 hours postoperatively ofrespiratory failure while suffering from a Corona virus infection. The 7surviving rats (3 male/4 female) were available for follow-up.

In female rats, the grafted bladder was tested for leakage byinstillation of saline through a urethral tube. Because atraumaticcatheterization is extremely difficult in male rats, the bladder wasfilled with a 27-gauge hypodermic needle through the posterior hostbladder wall. When satisfactory closure was achieved, the abdominal walland the skin were closed in two layers, with 4-0 plain gut (Davis+Geck)and 3-0 braided silk (Ethicon), respectively. No drainage was used andno drugs were administered peri- and postoperatively.

All animals were sacrificed 4 months after surgery. Two bladders of eachgroup were saved for histologic evaluation only. The bladders werefilled with 10% formalin after ligation of both ureters and urethra,thus allowing fixation in a distended state. The remaining bladders weresubjected to immediate tissue bath studies and histologic evaluationthereafter.

Only 29% of the control rats presented with the formation of 1 to 7bladder stones, in contrast to almost 85% of the grafted animals. Stoneformation was especially pronounced in the hamster BAMG-grafted animals(6 to 32 calculi). However, the composition of the calculi (struvite,60-100%; apatite, 40-100%; Newberryite, 20-100%; and brushite, 10-100%)did not differ among the control and the grafted groups.

2.3 Evaluation of Micturition Pattern

Volumes per void were evaluated non-invasively in all animalspreoperatively and at 3 and 7 days and 1, 2, 3 and 4 months aftersurgery. A specially designed “micturition cage” was used. The cageconsisted of four standard “hanging-basket” housing cages (10×8 inches)with a 0.5-inch metal grid bottom. These were placed on wooden supports(space maintainers) on a rack featuring a siliconized ⅛-inch hardwarecloth, which effectively prevented stool particles from falling into thecollecting trays while not hindering free passage of urine. Thecollecting trays were made of waste X-ray films chosen because thematerial is stiff, lightweight and water-repellent. The trays coveredthe entire area under each cage and were suspended by four nylon stringsto isometric force displacement transducers (Omega Engineering Inc.,Stamford Conn.) whose range of detection was 0.1 to 65 mg of urine. AnSCII-1121 signal processor (National Instruments Corp., Austin Tex.)supplied the excitation voltage to the transducers and fed the analogforce data to an NB-MIO-16 analogto-digital converter (NationalInstruments). A Macintosh Quadra 800 personal computer was used for dataacquisition. With the Laboratory Virtual Instrument EngineeringWorkbench software program (LabView®, National Instruments), a 4-channelvirtual instrument was designed to collect and display the digitalizeddata and to save it in a spreadsheet format transferable to theMicrosoft Excel software program, thus facilitating graphic andstatistical analysis.

Because approximately two-thirds of all voids in rats occur at night(Eika, et al. J. Urol. 151:496-502 (1994)), urinary excretion, voidingfrequency and volumes per void were studied for 12 hours overnight. Allrats were allowed to equilibrate in the housing cages for 8 to 10 hourswith food and water given ad libitum. To enhance diuresis, no food but asweetened orange-flavored instant drink (Tang™, Kraft General FoodsInc., White Plains N.Y.) was offered during the 12-hour study period.

Although mean preoperative volumes per void were slightly higher in thepartial cystectomy group, this difference was not statisticallysignificant (p=0.055 vs. the hamster BAMG group). Three days aftergrafting, volumes per void were decreased by more than 50% in allanimals. Within 2 months they were gradually restored; at the end of the4-month observation period, they exceeded preoperative values by 94% inthe grafted animals and 35 % in the control group. The difference fromcontrol in absolute volume per void was not statistically significant,however (dog BAMG, p=0.061; hamster BAMG, p=0.458; rabbit BAMG, p=0.159)(Table 2).

TABLE 2 Volumes per Void After Partial Cystectomy and inBAMG-regenerated Rat Bladders Within 4 Months of Surgery Hamster DogRabbit Partial BAMG BAMG BAMG Cystectomy (n = 5) (n = 5) (n = 5) (n = 7)Pre-operative 0.47 ± 0.06 0.57 ± 0.07 0.51 ± 0.05 0.65 ± 0.04 3 Dayspostop. 0.25 ± 0.01 0.37 ± 0.03 0.28 ± 0.02 0.34 ± 0.02 (−53%)* (−64%)(−55%) (−52%) 1 Week postop. 0.37 ± 0.07 0.48 ± 0.03 0.36 ± 0.01 0.47 ±0.02 1 Month 0.41 ± 0.07 0.67 ± 0.05 0.66 ± 0.03 0.64 ± 0.04 postop. 2Months 0.63 ± 0.09 0.72 ± 0.04 0.71 ± 0.12 0.80 ± 0.06 postop. 3 Months0.76 ± 0.08 0.99 ± 0.12 1.04 ± 0.15 0.82 ± 0.05 postop. 4 Months 0.81 ±0.11 1.08 ± 0.10 0.99 ± 0.06 0.88 ± 0.06 postop. (+72%) (+90%) (+94)(+35%) All data are expressed as mean value ± S.E.M. *Percentages ofpreoperative values are given in italics.

Fluid consumption and consequent urine excretion increased 2.2-fold inthe grafted and 1.3-fold in the control animals during the 4-monthobservation period. Voiding frequency initially increased by 42% in thegrafted and 77% in the control group. At 4 months after surgery, voidingfrequency was almost the same as pre-operatively in all groups.

2.4 Cystometric Measurements

Cystometric evaluation was performed at 4 months according to a methodmodified from Malmgren et al. (Malmgren, et al. J. Urol. 137:1291-1294(1987)) and Dorr (Dörr, J. Urol. 148:183-187 (1992)). All rats wereanesthetized with urethane (1.100 mg/kg i.p.) and placed supine on awarmed operating table. In female rats, transurethral cystometry wasperformed by means of a 24-gauge angiocatheter connected by polyurethanetubing (PE-90) to a pressure transducer (Baxter Uniflow pressuretransducer, Baxter Healthcare Corp., Irvine Calif.). An SCXI-1121 signalprocessor (National Instruments Corp., Austin Tex.) supplied theexcitation voltage to the transducer and fed the analog pressure data toan SCXI-1000 analog-to-digital converter (National Instruments). AMacintosh Quadra 800 personal computer with the LabView® softwareprogram (National Instruments) was used to acquire data and save it inspreadsheet format. In male rats open cystometry was performed. ThePE-90 tubing with a cuff at its tip was inserted through a smallincision in the lower ventral bladder wall where it was fixed with a 7-0Dermalon™ tobacco sac suture. Before the experiments were begun, thepressure transducers with tubing and angiocatheter attached were zeroedto the atmosphere during infusion. The bladder was emptied before eachcystometric measurement. After an equilibration period of 15 minutes,each rat underwent five consecutive cystometric measurements duringinfusion of warmed saline (37° C.) at 0.2 mL per minute with a HarvardApparatus 22 pump (Harvard Apparatus, Millis Mass.). Upon infusion,capacity was determined as the volume at which any kind of leakageoccurred. Baseline pressures, bladder opening pressures and peakpressures were measured. Compliance (cm H₂O/mL) was calculated accordingto the formula: $\frac{P_{2} - P_{1}}{V_{2} - V_{1}}$

with P₂ representing the bladder opening pressure and V₂ the infusedvolume at that time and P₁ representing the baseline pressure and V₁ theinfused volume at that time. Hence, high values reflect poor compliance,while small values characterize good bladder compliance.

Slight differences in baseline pressure proved not to be statisticallysignificant (Table 3).

TABLE 3 Cystometric Findings After Partial Cystectomy and inBAMG-regenerated Rat Bladders 4 Months Postoperatively Base- Bladderline Opening Peak Ca- Pressure Pressure Pressure pacity Compliance(cmH₂O (cmH₂O) (cmH₂O (ml) (cmH2O/ml) Hamster 4.4 21.2 43.3 2.49* +11.5* + (n = 5) ± 0.4 ± 1.8 ± 3.0 ± 0.04 ± 1.6 BAMG Dog (n = 5) 4.1 19.234.3 2.23* + 8.8* BAMG ± 0.9 ± 1.2 ± 2.8 ± 0.09 ± 0.8 Rabbit (n = 5) 2.320.8 41.8 2.08* + 9.9* BAMG ± 0.8 ± 0.8 ± 0.7 ± 0.04 ± 1.6 Partial (n =7) 3.7 19.3 47.4 1.43 24.8 Cystectomy ± 2.2 ± 1.5 ± 2. ± 0.08 ± 1.0 Alldata are expressed as mean value ± S.E.M. *significant difference (p <0.05) when compared with the partial cystectomy group. +significantdifference (p < 0.05) when compared with other BAMG-regeneratedbladders.

The bladder opening pressure was almost identical in all groups. Thepeak pressure was the highest in the partial cystectomy group, followedby the hamster and rabbit BAMG groups, with the lowest values in the dogBAMG-regenerated bladders; these differences were not statisticallysignificant (p≧0.154). In contrast, bladder capacity was significantlyhigher than the control in all grafted animals (hamster BAMG,p=3.39×10⁻⁷; dog BAMG, p=2.10×10⁻⁵; rabbit BAMG, p=0.012). Thedifferences in bladder capacity among the grafted groups were alsostatistically significant (hamster vs. dog BAMG, p=0.027; dog vs. rabbitBAMG, p=3.66×10⁻⁷). Compliance was significantly decreased in thepartial cystectomy group (p<1.96×10⁻⁴). Among the grafted groups, thehamster BAMG-regenerated bladders were significantly less compliant thanthe dog BAMG-regenerated bladders (p=0.03). Bladder calculi might haveinterfered with the cystometric measurements to varying degrees.Furthermore, bladder hypertrophy consequent to excessive stone formationmight have adversely affected compliance in the hamster BAMG-regeneratedbladders.

2.5 Tissue Bath Experiments

Tissue Preparation: The excised bladders were placed in a Petri dishwith chilled Krebs solution and carefully dissected free from theadherent connective tissue. Muscle strips of the same size (2×7 mm) ofthe regenerated bladder dome and the host bladder side wall wereobtained. These were mounted in the tissue bath to a glass tissuesupport hook (Radnoti Glass Technology, Monrovia Calif.) on one side andan isometric force displacement transducer, (Radnoti Glass Technology)on the other by means of two spring-wire clips connected with 4-0braided silk. All preparations were sufficiently durable to withstandthe grip of the wire clips during repeated or continuous contractions.

Bladder stone formation is a common finding after lower urinary tractsurgery in the rat. Guan, et al. J. Urol. 461-5; discussion 474 (1990);Kropp, Urology 46:396-400 (1995); Liang, et al. Invest. Urol. 12:5-7(1974); Little, et al. J. Urol. 152:720-724 (1994); Vaught, et al. J.Urol. 155:374-8 (1996). Indeed, this was true for the majority (85%) ofgrafted animals in our study. Bladder calculi may eventually causechronic inflammation and/or hypertrophy and dilation of the bladderthrough outlet obstruction, potentially affecting not only the in vivovoiding characteristics but also the in vitro contractility. To ensurethe same starting point for our organ bath experiments, we compared theBAMG-regenerated smooth muscle strips to strips from the same hostbladder wall rather than to bladder strips from untreated agematchedcontrol rats. Although all strips were cut about the same size, theregenerated and host bladder strip weights varied considerably.Differences in muscularization and collagen content and/or bladderhypertrophy are possible explanations for this phenomenon. The peakcontraction values (Table 4) were therefore related to the weight ofeach individual muscle strip.

TABLE 4 Organ Bath Findings in BAMG-Regenerated Rat Bladder Strips 4Months After Grafting Maximum Forces of Contraction Electrical FieldCarbachol High Strip Resting Potassium Weight Tension (mNewton/mg ofbladder (mg) (gm) wall tissue) Hamster Matrix 47.6 + 0.78 0.26* 0.59*0.12 BAMG (n = 4) ± 4.2 ± 0.03 ± 0.07 ± 0.01 ± 0.02 Strips Host 37.80.73 0.62 1.19 0.41 (n = 10) ± 2.5 ± 0.08 ± 0.12 ± 0.22 ± 0.11 DogMatrix 34.3 0.9 0.34 0.61 0.31 BAMG (n = 4) ± 3.6 ± 0.01 ± 0.02 ± 0.02 ±0.01 Strips Host 53.6 0.82 0.40 1.091 0.36 (n = 10) ± 5.6 ± 0.05 ± 0.09± 0.19 ± 0.10 Rabbit Matrix 23.4 0.78 0.30* 0.61* 0.20 BAMG (n = 4) ±1.1 ± 0.08 ± 0.05 ± 0.14 ± 0.03 Strips Host 34.0 0.71 0.80 1.79 0.45 (n= 10) ± 3.6 ± 0.07 ± 0.06 ± 0.16 ± 0.12 All data are expressed as meanvalue ± S.E.M. *significant difference (p < 0.05) when compared withhost bladder smooth muscle strips. +significant difference (p < 0.05)when compared with other BAMG regenerate strips.

Tissue Bath and Recording and Stimulating Equipment: The 30-mLdouble-chambered Quiet Bath (Radnoti Glass Technology) was used. Itsworking chamber was connected to a second chamber with 95% oxygen and 5%CO₂ infusion. Gas flow induced circulation of the Krebs solution, whichwas warmed to 37° C. by an external heating circuit (circulating pumpThermomix BU, Braun Instruments, Burlingame Calif.). Forces lower than 1mNewton (0.1 g) without bubble artifacts could be measured in theworking chamber. The transducer signals were fed into a thermal arrayrecorder (Gould TA 4000, Gould Inc., Valley View Ohio). For tissuestimulation, vertical L-shaped, custom-made platinum iridium electrodes(15 mm long, 0.18 mm diameter) separated by 10 mm were used with a GrassS44 stimulator (Grass Instrument Co., Quincy Mass.). The tissue wasmounted parallel to the electrodes at a preload of 20 mNewton (2 g)resulting in a resting tension of about 4 mNewton (0.4 g) at the end ofa 60-min equilibration period. A custom-made current-distribution boxsupplied a supramaximal current of 0.14 A at 15 V, which wassequentially delivered to two of eight chambers.

Electrical Field Stimulation: On the basis of preliminarylength-tension, frequency-response and current-response studies ofnormal rat detrusor smooth muscle, we used the following supramaximalstimulation parameters: bipolar, monophasic balance-charged rectangularpulses; 1 msec pulse duration; 1-80. pulses per second (pps) frequency;10 sec stimulation trains; 2 min intervals between stimulations; 0.14 Aamplitude at 15 V.

Electrical field stimulation was used to evaluate nerve-mediated smoothmuscle contraction. Carbachol (as the classic cholinergic agonist) andatropine (as the appropriate antagonist) were used to demonstratefunctionally the presence of cholinergic receptors within theregenerated tissue. Potassium was used as another mechanism to inducesmooth muscle cell depolarization through a direct chemical change ofthe membrane potential.

Supramaximal electrical field stimulation at increasing frequenciesshowed contractile responses in all BAMG-regenerated strips that werequalitatively very similar to the host bladder smooth muscle strips.Peak contractions uniformly occurred at a stimulation frequency of 40pulses per second in both the regenerated and the host bladder strips(FIG. 3). The maximal force of contraction was almost the same in allBAMG-regenerated strips and amounted to 85% (dog BAMG), 42% (hamsterBAMG) and 38% (rabbit BAMG) of the appropriate host bladder wall tissue(Table 4).

Carbachol stimulation (1×10⁻⁴M) also elicited a qualitatively identicalcontraction in the BAMG and host bladder strips. Again, the maximalforce of contraction was the same in all BAMG-regenerated strips. Thisforce amounted to 60% (dog BAMG), 50% (hamster BAMG) and 34% (rabbitBAMG) of the appropriate host bladder wall tissue and it was on averagetwo-fold higher than the peak contractions evoked by electricalstimulation. Administration of atropine (1×10⁻⁶ M) completely relaxedall carbachol-contracted muscle strips even below their initial restingtension, also diminishing the response to subsequent electricalstimulation by more than 90% in all strips.

2.6 Solutions and Drugs

Krebs (mmol): NaCl 118.1, KCl 4.6, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25.0,CaCl₂ 2.5, glucose 11.0. In “high potassium” Krebs buffer solution (KCl60 mM), NaCl was replaced with equimolar amounts of KCl. All solutionswere made fresh from stock solutions. Carbachol (Sigma Chemical Co., St.Louis Mo.; C 4382) and atropine sulfate (Sigma A 0257) were diluted insaline. The amounts and volumes added to the bath were as follows:Carbachol 1×10⁻⁴M, 1 mL; Atropine 1×10⁻⁶M, 1 ML.

High-potassium (60 mmol) Krebs buffer solution caused sustainedsubmaximal contraction in both the BAMG-regenerated and host bladdersmooth muscle strips. The differences in maximal force of contraction inthe grafted group were not statistically significant. Once more, peakcontractions were smaller in the regenerated strips. They amounted to86% (dog BAMG), 44% (rabbit BAMG) and 29% (hamster BAMG) of theappropriate host bladder wall tissue (Table 4).

2.7 Staining

After fixation with 10% formalin for at least 24 hours, the specimensfor light microscopic examination were embedded in paraffin, sectionedand stained with trichrome for collagen and smooth muscle, hematoxylinand eosin (H&E) for nuclei, α-actin for smooth muscle and urothelium,and protein gene product (PGP) for nerves. Specimens were also preparedfor scanning electron microscopy.

Macroscopically, the inflated bladders were uniformly dilated withoutevidence of diverticuli formation in the region of the graft. Mild tomoderate adhesions to the surrounding tissue were found in all animals,including the partial cystectomy group, next to the identificationsutures rather than within the matrix area itself. Within 4 months aftersurgery there seemed to be a reversal of the ratio of matrix area/hostbladder area, suggesting significant autoregeneration of the hostbladder and relative shrinkage of the BAMG. No macroscopic signs ofhydronephrosis or other upper tract deterioration were noted atsacrifice in any of the animals.

Histologic staining of all dog, hamster and rabbit BAMG-regeneratedbladders showed a bladder wall structure that was qualitativelyidentical to the host bladder because all three layers of normal ratbladder were present. The inner surface of the grafts was covered by auniform urothelial lining with a differentiated muscularis mucosas,although most bladders had global urothelial hyperplasia resulting fromstone formation. Distinct bundles of well developed, spatially orienteddetrusor smooth musde were evident throughout the grafted area. Thethickness of these muscle bundles seemed to decrease in the central partof the BAMGs, however. The number of well-formed blood vessels (small-and large-diameter) appeared greater in the BAMG-regenerated than in thehost bladders. The opposite was true for PGP-positive nerve fibers.There were no mononuclear inflammatory cell infiltrates or otherhistologic signs of rejection in any of the BAMGs.

2.8 Statistics

A two-tailed Student's t-test was used to compare volumes per void,cystometric values and organ bath data in the control and graftedanimals; p values <0.05 were considered statistically significant. Alldata are presented as mean ±S.E.M.

What is claimed is:
 1. An insoluble elastic matrix graft for repairingbladder smooth muscle having the following properties: (i) the matrixgraft is derived from bladder smooth muscle tissue; (ii) the matrixgraft is impermeable to urine; (iii) the matrix graft consistsessentially of an intact framework of collagen and elastic fibers thatis free of cell contents; and, (iv) the framework permits growth ofmuscle cells within the framework.
 2. A matrix graft in accordance withclaim 1, said matrix graft has the shape of a membrane patch.
 3. Amatrix graft in accordance with claim 1, said matrix graft beingprepared from tissue isolated from an animal selected from the groupconsisting of rat, rabbit, hampster, dog, pig and human.
 4. A matrixgraft in accordance with claim 1, said matrix graft being prepared fromtissue isolated from an animal selected from the group consisting ofrat, rabbit, hampster, dog, pig and human, and indicating essentially nocell nuclei when stained with a dye selected from the group consistingof trichrome, H&E, α-actin and PGP.
 5. A matrix graft in accordance withclaim 1, said matrix graft being isolated from human bladder tissue andhaving an elastic modulus of about 0.40 to about 0.80 MPa.
 6. A matrixgraft in accordance with claim 1, said matrix graft being isolated fromrat bladder tissue and having an elastic modulus of about 0.80 to about2.10 MPa.
 7. A matrix graft in accordance with claim 1, said matrixgraft being isolated from pig bladder tissue and having an elasticmodulus of about 0.25 to about 0.60 MPa.
 8. A matrix graft of claim 1wherein the matrix graft is prepared using a combination of exogenousdetergents and enzymes.
 9. A matrix graft of claim 1 wherein theelasticity of the matrix graft is between 0.25 MPa and 2.10 MPa.
 10. Amatrix graft of claim 1 wherein the matrix is treated with a nuclease toremove nucleic acid.